Lab Tools for Viral Vector Gene Delivery
Viral vector gene delivery is one of the most widely used methods to insert foreign genetic material into target cells. In chimeric antigen receptor (CAR) T-cell therapy, lentivirus vectors are the method of choice as they offer high efficiency, stability and versatility of gene transfer.
However, there are challenges in the process that need to be addressed, like optimization of transduction efficiency, purification and characterization of the critical quality attributes of the vectors.
Sartorius offers orthogonal technologies to streamline R&D and lab-scale production of lentiviral vectors:
- Novel, rapid image-based assay for infectious titer determination
- Rapid, one-step harvest and clarification of lentiviral vectors
- Development of packaging cell lines for lentivirus production
Incucyte® Live-Cell Analysis Platform
Continuous, real-time live-cell imaging and analysis platform performs analysis without removing cells from the incubator or disturbing cultures.
- Profile type-specific and time-dependent biological activity of cells
- Visualize and validate results with cell images and movies
- Enjoy walk-away convenience as cell images are automatically acquired and analyzed
Featured application: Rapid and accurate determination of lentivirus infectious titers
Functional infectious titer determinations of viral vector samples are of major importance with vector quality control being a significant bottleneck for viral vector process development and production.
We developed a novel, real-time imaged-based approach to quantify the infectious titer of anti-CD19 CAR lentiviral vectors using the Incucyte® Live-Cell Analysis System.
Related Application Note: Real-Time Live-Cell Analysis of Lentiviral Titer Determination
Explore Live-Cell Analysis Instruments
Sartoclear ® Dynamics Lab
A single-step method for harvesting mammalian cell cultures with high cell densities. It utilizes membrane filtration with the use of diatomaceous earth (DE) addressing the challenges of the CAR-T workflow due to:
- Rapid filtration
- Safe handling
- Preservation of viral activity
Featured application: Lab-Scale Harvest and Clarification of Lentiviral Vectors
Efficient lentivirus harvesting method to eliminate major contaminants and to reduce turbidity of the solution, while maintaining vector activity critical in CAR-T workflows. The method provides the highest recoveries of LV particles and infectious titers.
Lentiviral vectors were produced by transient transfection of HEK293T/17 SF suspension cells and clarification was carried out with Sartolab® RF50 and Sartoclear Dynamics® Lab V50.
Related Application Note: Lab-Scale Harvest and Clarification of Lentiviral Vectors Using Sartoclear
Kits and IEX Units for Virus Purification and Plasmid DNA Isolation
Vivapure® Lentiselect and Adenopack
Lentivirus and Adenovirus purification kits utilize membrane chromatography for fast and safe virus purification from 20 to 1,000 mL cell cultures. High yields with low endotoxin levels are ideal for downstream applications such as cell-based assays.
- Ready-to-use kits with buffers and consumables needed for purification
- Purify and concentrate viral vectors in only a few hours
- No need for time-consuming ultracentrifugation
Sartobind® Lab or Vivapure® D Units
For isolation and purification of plasmids from host cell proteins, RNA and chromosomal DNA.
Ion exchange on this membrane chromatography units enables rapid isolation of plasmid DNA to sufficient purity for restriction enzyme digests and sequencing
CellCelector Automated Cell Selection and Retrieval Platform
A fully automated cell imaging and picking system developed for detection, selection and isolation of single cells, clusters, spheroids and organoids as well as single cell clones and adherent colonies.
One-round single cell cloning and cell line development for lentivirus packaging and production cell lines with integrated monoclonality proof.
The CellCelector is the ideal solution for the automated selection and isolation of bacterial colonies with the semi-solid media picking module which is developed for picking cell colonies from semi-liquid medium (e.g. Methylcellulose) and from solid media (e.g. Agar).
Related publication:
Broussau, S., Lytvyn V., Simoneau M. et al:
Packaging cells for lentiviral vectors generated using the cumate and coumermycin gene induction systems and nanowell single-cell cloning. Molecular Therapy: Methods & Clinical Development Vol. 29 June 2023 57
Benefits for CAR-Vector Preparation and Testing
Sartoclear Dynamics® Lab V50 and Sartolab® RF50 filtration units enable clarification of lentivirus-producing HEK293T cultures in a single step, eliminating the centrifugation step and reducing processing time. This method also achieved the best results in lentiviral particle recovery at 87%.
Incucyte® Live-Cell Analysis Systems offer rapid, non-invasive, and simplified approach for high-throughput functional quantification of infectious titer of anti-CD19 CAR lentiviral vectors. Real-time, kinetic readouts and integrated software enable tracking of viable transduced cells over time and allow determination of optimal readout timepoints.
Sartoclear Dynamics® Lab mammalian cell culture clarification and Vivapure® membrane chromatography plasmid DNA - as well as lentivirus purification and concentration kits - offer researchers fast, safe and easy to use solution. Select the most convenient kit for your sample volumes!
Resources for CAR-Vector Preparation and Testing
Frequently Asked Questions
Utility of the Incucyte® Titer Assay was illustrated in a stability study examining the effect of a range of physical conditions virus infectivity. LV titer was maintained over four freeze-thaw cycles enabling storage of LV between DSP steps at -80 ºC for future analytical testing. Investigating which process parameters affect infectious titers is important to ensuring the infectivity potential of viral vectors is maintained.
The use of Sartoclear Dynamics® Lab V50 reduces the handling time as it eliminates the need for a centrifugation step and therefore facilitates fast handling. Additionally, Sartolab® RF50 features an excellent filter device design that enables safe handling by minimizing the risk of spillage of the virus material, a large filter area. A multi-parallel usage is possible together with the MultiStation and it provides the best results in lentiviral particle recovery at 87%.
Lentivirus purification and concentration kits offer researchers who need to recover up to 4 – 5 × 109/mL purified recombinant lentivirus particles for in vitro transfection a fast, safe and easy to use solution.
Three kit sizes are available for the purification and concentration of VSV-G pseudotyped lentivirus, from 40 mL, 500 mL or up to 1 L cell cultures, yielding high titers of 8 × 108 to 1 × 1010 infective particles.
To reach a high throughput and optimal yields in the bacterial cell line development automation of the production process is of great interest. The CellCelector with the semi-solid media picking module - developed for picking cell colonies from semi liquid medium (e.g. Methylcellulose) and from solid media (e.g. Agar) enables the automated selection and isolation of positive bacterial colonies for evaluation and further downstream processing.
